Abstract

BACKGROUND:

Fluorescence and bioluminescence resonance energy transfer (F/BRET) are two forms of Förster resonance energy transfer, which can be used for optical transduction of biosensors. BRET has several advantages over fluorescence-based technologies because it does not require an external light source. There would be benefits in combining BRET transduction with microfluidics but the low luminance of BRET has made this challenging until now.

METHODOLOGY:

We used a thrombin bioprobe based on a form of BRET (BRET(H)), which uses the BRET(1) substrate, native coelenterazine, with the typical BRET(2) donor and acceptor proteins linked by a thrombin target peptide. The microfluidic assay was carried out in a Y-shaped microfluidic network. The dependence of the BRET(H) ratio on the measurement location, flow rate and bioprobe concentration was quantified. Results were compared with the same bioprobe in a static microwell plate assay.

PRINCIPAL FINDINGS:

The BRET(H) thrombin bioprobe has a lower limit of detection (LOD) than previously reported for the equivalent BRET(1)-based version but it is substantially brighter than the BRET(2) version. The normalised BRET(H) ratio of the bioprobe changed 32% following complete cleavage by thrombin and 31% in the microfluidic format. The LOD for thrombin in the microfluidic format was 27 pM, compared with an LOD of 310 pM, using the same bioprobe in a static microwell assay, and two orders of magnitude lower than reported for other microfluidic chip-based protease assays.

CONCLUSIONS:

These data demonstrate that BRET based microfluidic assays are feasible and that BRET(H) provides a useful test bed for optimising BRET-based microfluidics. This approach may be convenient for a wide range of applications requiring sensitive detection and/or quantification of chemical or biological analytes.

All treatments included 1 µM GFP2-RG-RLuc. The control was 1 µM RLuc, 1 µM GFP2 and 5 µM native coelenterazine. Thrombin pre-treatment was 54 nM thrombin for 90 minutes at 30°C. The thrombin+Hirudin condition involved addition of 2 units of hirudin at room temperature 10 minutes before addition of 54 nM thrombin. All data were measured in a 96 well microplate using a SpectraMax M2 spectrofluorometer.

Variation in luminescence with distance along the common microfluidic channel.

GFP2-RG-RLuc luminescence intensities (AU) for RLuc (+) and GFP2 (▿) channels in the absence of and for RLuc (○) and GFP2 (×) channels in the presence of coelenterazine substrate. (♦) BRETH ratio as a function of distance x from the Y-junction as labelled in . The concentration of GFP2-RG-RLuc was 3.0 µM and the concentration of coelenterazine was 58.6 µM. [The higher substrate concentrations used in microchip is to increase the light output from the much smaller volume.] Flow rate in the common channel was 40 µl/h. Other conditions as described in “Experimental” and .

For both static and microfluidic assays, BRETH ratios were normalised independently against the ratio measured in the absence of thrombin. All microfluidic measurements were obtained at x = 2.1 mm. (○) microfluidic and (□) multiwell plate measurements. The large graph shows the BRETH responses at low thrombin concentrations. Insets show the corresponding full-range measurements. Data are fitted to linear regressions: y = 0.835x +1.019 (R2 = 0.996) for the microfluidic and y = 0.1797x +1.001 (R2 = 0.995) for the multiwall.